In the scope of biotechnology, there are produced biological products, for example monoclonal antibodies, interferons, vaccines, plasminogen activators and the like, with the help of cells and especially of mammalian cells. However, the economics of such processes depend essentially upon the cells multiplying as much as possible and upon the highest possible concentration of the desired substance being produced in the interior of the cells. In order to achieve these objects, special requirements are demanded of the processes and devices used for the large scale culturing of cells. Thus, in particular, provision must be made for a sufficient supply of nutrients, as well as of gases, particularly oxygen. Waste products, i.e. cell metabolic products, which are not needed in the cell culture and which frequently inhibit growth of the cells, must be removed. All other environmental conditions, for example temperature and pH value, must also be as optimal as possible.
In an article entitled "Mammalian cell culture: engineering principles and scale-up" by M. W. Glacken et al. in the periodical "Trends in Biotechnology", pp. 102-108/1983, there are discussed these problems and a number of constructional principles for appropriate apparatus for the culturing of cells.
As can be seen from this article, it is necessary to differentiate between suspension cells and carrier-dependent cells. Whereas the suspension cells float freely in the cell culture medium and can grow therein, carrier-dependent cells, which are also called adherent cells, require a solid carrier in order to be able to survive and grow.
In this article, besides the above-mentioned problems, there are, in particular, discussed the problems of "upscaling". Thus, it has been found that methods which function adequately on a laboratory scale are unsuitable for large-scale production.
Traditionally, mammalian cells, as well as bacterial cells, are primarily cultured as suspension cultures in bioreactors which are also called fermenters. The environmental conditions can be precisely controlled in such vessels. A stirring means moves the culture medium in the interior of the reactor and thus provides for a homogeneous distribution of the cells. A corresponding culture technique is also possible for adherent cells in that they are present on small carrier spheroids which are referred to as "micro-carriers". These float in the culture medium.
The supply of nutrients to the cells and the removal of waste materials takes place, in the case of suspension cultures in bioreactors, according to one of the following three processes:
In a batch operation, the reactor is operated discontinuously. At the beginning of a batch, the culture medium usually contains a serum, for example fetal calf serum (FCS), and the necessary nutrients, for example glucose, vitamins, amino acids and minerals. In operation, these are consumed so that the medium becomes more and more impoverished in nutrients. At the same time, the concentration of waste products increases which finally results in a prevention of the cell growth. The result of this is a course of cell density curve such as is shown in FIG. 1a of the accompanying drawings. The cell density achieves a maximum value of about 10.sup.6 cells/ml. and thereafter decreases again. Consequently, the culturing is discontinued when the maximum cell density is achieved. The contents of the reactor is then passed on for further working up. This process is unsatisfactory insofar as the environment of the cells changes continuously so that, during most of the time of the fermentation procedure, it is by no means optimal.
This could be improved by repeatedly refreshing the culture medium without thereby removing cells. However, for this purpose, a part of the culture medium must be repeatedly removed even though it has by no means been consumed. Such a process is extremely expensive because the known culture media are difficult to obtain and, consequently, are expensive.
Better in this regard is the so-called "feedbatch process" in which, during the fermentation procedure, fresh culture medium is not supplied in its totality but only the consumed nutrients are continuously supplied. However, in practice this process does not provide any substantial advantages because the increase of the waste materials leads to a characteristic course of the cell density during the culturing procedure similar to that in the case of the purely batch process.
The third process is the continuous process in a so-called chemostat or cytostat. Here, the environmental conditions can be uniformly adjusted so that the cells can grow optimally. However, the process is very laborious and expensive because culture medium must be continuously supplied and removed. Furthermore, in the case of this process too, there is not achieved a substantially higher cell density than in the case of the above-mentioned processes because cells are also continuously removed from the reactor with the running off of cell culture medium.
Regardless of which of the three described operational processes is chosen for the suspension cultures, the results are not satisfactory especially because the consumption of valuable culture medium is too high and because a higher maximum cell density is desirable. This latter criterion is of especial importance because present experience shows that the concentration of the desired products in the cells increases with increasing cell density. Therefore, an increase of the cell density leads to an overproportional increase of the yield of product.
In order to achieve a further improvement, new processes have been suggested.
The so-called "systems with artificial capillaries" consist essentially of a bundle of hollow dialysis fibres which are arranged in the interior of a hollow cylinder. They are especially recommended for the culturing of adherent cells. The cells are thereby applied to the outer side of the hollow fibres and are present in the intermediate spaces between the hollow fibres within the cylindrical housing. A culture medium flows through the inner side of the capillaries and air is supplied to the cells through the outer side of the housing. With such a process, there is achieved a comparatively uniform and economic supply of the nutrients to the cells. The cell density can also be slightly increased. Although the supply of nutrients is uniform, nevertheless it has been shown that the cells are supplied to a varying extent. This is especially due to the fact that they are positioned on the capillaries in several layers. The supply of oxygen is thereby also limited which can result in an increased production of waste products.
Such hollow fibre fermenters are described in European Patent Specifications Nos. 0,112,154 and 0,112,155. In the latter Specification, the system is varied insofar as the cells are pumped by means of a pump in circulation through the culturing module.
Hollow fibers, the walls of which consist of a membrane material, are also used in the cell culturing process described in published Japanese Patent Specification No. 60-207581. The hollow fibers are present in the culture medium in the inner chamber of a fermenter. The inside of the hollow fibers is connected with the outside of the fermenter via a single pipe which serves not only for the supply but also for the removal of liquids. With the help of a pump, waste materials of the cells are sucked off through this pipe through the wall of the hollow fibres or, in a cycle chronologically separate therefrom, sterilized water, which does not harm the cells, is introduced. Nutrients for cells can possibly also be added to this sterilized water. In operation, pumping into the culture liquid and sucking out therefrom is carried out alternatingly. This cycle is repeated continuously. In order to avoid the disadvantages of this discontinuous method of operation, there is preferably used an even number of hollow fibre bundles which are operated alternatingly, i.e. while sucking off from the culture medium through one half of the hollow fibre bundles, liquid is introduced through the other half of the fibre bundles. This process is comparatively laborious and involves the danger that the hollow fibres are damaged by the continuously alternating pressure loading from one side to the other.
Another novel technique involves the enclosure of the cells in spheroidal microbodies made of a semipermeable membrane material. This is indicated in the cited article as the sole new process for the culturing of suspension cells. It leads to an increase of the cell density in the small spheroidal bodies which, in turn, results in an increase of the product yield. The harvesting of the cells is simple because the spheroidal bodies settle out due to gravity, whereas individual cells must, as a rule, be centrifuged. These advantages are countered by the very high costs for the encapsulation of the cells. Furthermore, the supply of oxygen to the cells is limited which, in turn, reduces the success of the culturing.